DNA/RNA microarrays have become a leading method in gene analysis, medical diagnosis and disease prevention, in large measure due to the significant automation and high throughput they bring. Based on the hybridization between surface-immobilized probes and DNA/RNA targets, large amounts of genetic information can be extracted from one assay very efficiently. Many such assays use fluorescence-based detection techniques that typically require multiple steps to incorporate fluorophores into target sequences. An attractive alternative is to use a platform containing probes that become fluorescent upon binding to their target.
Molecular beacon probes are one such category of self-reporting probe (Tyagi et al. Nat. Biotechnol., 1996, 14, 303-308 and Tyagi et al. Nat. Biotechnol., 1998, 16, 49-53). However, the probes' signal-to-background ratio is often significantly reduced when molecular beacon probes are immobilized on solid substrates as needed in a microarray format. Several approaches have been used to decrease the background fluorescence and increase the hybridization of immobilized molecular beacon probes. Despite these efforts, the differences between molecular beacon probes immobilized on solid substrates and those in solution remain poorly understood. There is a need for surface-immobilized molecular beacon probes with high SBR ratios.